***MSP Primer Troubleshooting Specificity*** - (Dec/13/2007 )
I am new to this board, and this is my first topic. Apologies if this has been covered. I'll try not to throw out too many things at once. But if you have any questions please feel free to ask.
The problem I am having is with my MSP primers. They are designed to have two CpG in both the methylated specific and unmethylated specific. I am using a touchdown PCR program and the universally unmethylated samples are working on the methylated specific primer sets, and the universally methylated samples are working on the unmethylated specific primer sets.
I have tried to raise the annealing temps to make the PCR reaction more stringent, but I have been unsuccessful. I have primer designs for several genes, and would like to modify my PCR conditions to get favorable results.
Thanks in advance.
how was your universally methylated dna prepared?
sounds like it is not completely methylated if you have amplification using unmethylated primer sets.
Where are the CpG's in your primer? They will have little effect at the 5' end, and dramatic effect if located at or very near the 3' end. Ideally the 3' base would be selective for a converted or unconverted C. Also, remember that the reverse primer should have G's or A's, not C's or T's to amplify the converted/unconverted top strand.
^^methylnick, I used the methylseqr kit by AB to bisulfite convert. I ordered Universally Methylated/Unmethylated DNA from chemicon. I used the max concentration of 300ng as suggested by the kit.
Thanks phage^ they are located near the 3'end and I am using 2-3 CG's in my primer design.
I see your problem, it's known that the universally unmethylated DNA from chemicon is not as it says, in our hands.
We have performed tests by mixing methylated/unmethylated gDNA from the same company on sequenom assays to p16 and Rassf1 and found that there is residual methylation of around 10-20% in the UNMETHYLATED DNA and all our mixings were out by the same factor, we can only conclude that the unmethylated DNA is not unmethylated.
Use with caution, we have alliviated this by making our own unmethylated DNA from WGA.
Thank you for your help methylnick,
Going back to my previous problem of the methylated samples working on the unmethylated primer set; Do we agree that this is just a primer design problem? I've been thinking og designing the primers with three CpG islands and not including the last "G" in the primer design for higher specificity.
Any other suggestions?
Thanks and I hope everyone had a pleasant Holiday.
Has anyone had any experience with pyrosequencing? Can someone link me to a thread if it exists?